Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 328707, 12 pages http://dx.doi.org/10.1155/2015/328707 Research Article Wu-Tou Decoction Inhibits Chronic Inflammatory Pain in Mice: Participation of TRPV1 and TRPA1 Ion Channels Chao Wang,1 Chunfang Liu,1 Hongye Wan,1 Danhua Wang,1 Danni Sun,1 Tengfei Xu,2,3 Yue Yang,1 Yakun Qu,1 Ying Xu,1 Xianghong Jing,4 Junling Liu,4 Shuping Chen,4 Zhiqiang Liu,2 and Na Lin1 1 Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China 2National Center for Mass Spectrometry in Changchun & Jilin Province Key Laboratory for Traditional Chinese Medicine Chemistry and Mass Spectrometry & Chemical Biology Laboratory, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China 3University of Chinese Academy of Sciences, Beijing 100049, China 4Institute Acupuncture & Moxibustion, China Academy of Chinese Medical Sciences, Beijing 100700, China Correspondence should be addressed to Na Lin; [email protected] Received 5 January 2015; Accepted 20 February 2015 Academic Editor: Akio Hiura Copyright © 2015 Chao Wang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Wu-tou decoction (WTD) is a classic traditional Chinese medicine formula and has been used effectively to treat joint diseases clinically. Previous reports indicated that WTD possesses anti-inflammatory activity; however, its actions on pain have not been clarified. Here, we investigated the antinociceptive activity of WTD in CFA-induced mice, and its possible mechanism ofthe action associated with transient receptor potential (TRP) ion channels was also explored. Our results showed that 1.58, 3.15, and 6.30 g/kg WTD significantly attenuated mechanical, cold, and heat hypersensitivities. Moreover, WTD effectively inhibited spontaneous nociceptive responses to intraplantar injections of capsaicin and cinnamaldehyde, respectively. WTD also effectively suppressed jumping and wet-dog-shake behaviors to intraperitoneal injection of icilin. Additionally, WTD significantly reduced protein expression of TRPV1 and TRPA1 in dorsal root ganglia and skins of injured paw. Collectively, our data demonstrate firstly that WTD exerts antinociceptive activity in inflammatory conditions by attenuating mechanical, cold, and heat hypersensitivities. This antinociceptive effect may result in part from inhibiting the activities of TRPV1, TRPA1, and TRPM8, and the suppression of TRPV1 and TRPA1 protein by WTD was also highly effective. These findings suggest that WTD might be an attractive and suitable therapeutic agent for the management of chronic inflammatory pain. 1. Introduction that members of the transient receptor potential (TRP) ion channel family in nociceptor peripheral terminals and Chronic inflammatory pain ensues when the normal process dorsal root ganglions (DRGs) are fundamental molecules of inflammation does not resolve, resulting in excess proin- that detect noxious stimuli and transduce a diverse range flammatory cytokines and chemoattractants that can eventu- of physical and chemical energy into action potentials in ally lead to peripheral and central sensitization [1]. Indeed, somatosensory nociceptors and contribute to the initiation hypersensitivities to heat, cold, and mechanical stimuli are and maintenance of hypersensitivity under inflammatory well-documented characteristic symptoms of inflammatory and neuropathic pain conditions [5]. Among them, TRPV1, and neuropathic pain both in the clinical setting and in TRPA1,andTRPM8ionchannelshaveattractedthemost experimental animal models [2, 3]. It can be either recurrent attentions. It has been shown that deleting or inhibiting the or persistent and is a serious developmental health concern activities of these ion channels is associated with reduced that can interfere significantly with daily functioning4 [ ]. inflammatory nociceptive responses5 [ –7], and inflamma- Over the past decade, flourishing research has revealed tory hypersensitivity also appears to be dependent on the 2 BioMed Research International expression of TRPs [8–10]. Thus, pain-evoking TRP channels experiments. Take 1 mL and dilute it 1000 times as the analyte emerge as promising therapeutic targets for a wide variety of sample with internal standards. The sample was filtered pain and inflammatory conditions [5–7]. through a 0.22 m filter membrane and was quantified Treatment of chronic inflammatory pain still remains a by UPLC-MS/MS analysis. Standards of ephedrine, berber- major challenge in clinical practice because of its heteroge- ine, paeoniflorin, liquiritin, glycyrrhizic acid, calycosin-7- neous etiology and the complex underlying pathophysiology glucoside, formononetin, dioscin, and liquiritigenin were mechanisms.Themostcommonlyusedanalgesics,suchas obtained from the Chinese Authenticating Institute of Mate- opioids, nonsteroidal anti-inflammatory drugs (NSAIDs), rial and Biological Products (Beijing, China). Standards and anticonvulsants (for neuropathic pain), produce serious of benzoylmesaconine, benzoylhypacoitine, aconitine, and side effects [11, 12]. Therefore, there is an urgent need to benzoylaconitine were purchased from Lan Yuan Biological develop novel and safer pain medicines to meet the needs of Technology Co., Ltd. (Shanghai, China). The purity of all patients. In this context, natural products that present fewer compounds was more than 98% (determined by HPLC). side effects emerge as interesting therapeutic resources for They were prepared at a proper concentration in methanol. the development of new drugs for the management of certain HPLC-grade acetonitrile and formic acid were obtained from chronic pain states. Fisher Scientific (Loughborough, UK). Ultrapure water was Wu-tou decoction (WTD), a classic Traditional Chi- prepared by the Milli-Q plus (Milford, MA, USA) water nese Medicine (TCM) formula from Chinese medical sage purification system. Zhongjing Zhang, has been recorded in Jin Gui Yao Lue The UPLC-MS/MS system used was a Waters Acquity- for more than 1,000 years. It was formulated according to Ultra Performance LC system (Waters, USA) coupled with TCM theory especially for the treatment of cold pattern of Xevo TQ MS spectrometer with electrospray ion source (ESI) arthromyodynia. Clinically, WTD has been used effectively (Waters, Manchester, UK). Chromatographic separation was to treat joint diseases. Previous studies have shown that WTD performed on a Waters Acquity UPLC BEH C18 column ∘ possess the anti-inflammatory effect by inhibiting proinflam- (1.7 m, 2.1 ∗ 50 mm) kept at 35 C. Acetonitrile (A) and 0.1% matory cytokines such as IL-1 and TNF- and regulating aqueous formic acid (v/v) (B) were used as the mobile phase. TLR2/TRAF6/Faslg signal pathway in complete Freund’s The gradient elution with the flow rate of 0.3 mL/min was adjuvant- (CFA-) induced arthritis rats [13, 14]. However, it performed as follows: 10% A at 0–2 min, 10–15% A at 2–7 min, remains unclear whether WTD could inhibit inflammatory 15–30% A at 7–15 min, 30–39% A at 15–21 min, and 39% up to pain.Inthisstudy,weinvestigatedtheantinociceptiveactivity 100% A at 21–25 min. The sample injected volume was 5 L. ofWTDoncutaneousinflammationinducedbyCFAinmice, TheMSanalysiswascarriedoutbytheESIsourceinboth and its possible mechanism of the action associated with TRP positiveandnegativeionmode.Thesourcetemperaturewas ∘ ∘ ion channels was also explored. 110 C, and the desolvation gas temperature was 300 C. The flow rates of cone and desolvation gas were set at 30 L/h and 2. Materials and Methods 600 L/h, respectively. The voltages of capillary and extraction cone in positive ion mode were set at 2.5 kV and 5.0 V,respec- This study was supported by the Research Ethics Committee tively, and in negative ion mode, they were set at 2.0 KV and of China Academy of Chinese Medical Sciences, Beijing, 5.0 V, respectively. The multiple reaction monitoring (MRM) China (Permit number: 2013-61). All animals were treated in transitions for fourteen analytes as well as their respective accordance with the guidelines and regulations for the use cone voltages and collision energies are listed in Table 2. andcareofanimalsattheCenterforLaboratoryAnimalCare, China Academy of Chinese Medical Sciences. All efforts were 2.2. Analysis of Water Extract of WTD. The content of eleven made to demonstrate consistent effects of the drug treatments main components in WTD used in this trial was quantified by and minimize the suffering of animals. liquid chromatography-tandem mass spectrometry (UPLC- MS/MS), and the calibration curves, calibration ranges, and 2.1. Preparation of WTD. WTD is a mixture of five traditional content in 1 g WTD of these compounds are performed in drugs (Table 1). The drugs were purchased from Beijing Table 3. Huamiao Chinese Medicine Engineering Development Cen- ter (Beijing, China) and authenticated by Professor Shilin 2.3. Animals. MaleICRmice(30–35g)wereobtainedfrom Hu, China Academy of Chinese Medical Sciences. WTD was Laboratory Animal Center of Academy of Military Medical prepared according to our previous studies [14, 15]. All the Sciences, Beijing, China (License number: SCXK 2012-0004). They were kept in a temperature controlled environment components of WTD were dried and homogenized to fine ∘ powders; then the 84 g powdered materials were immersed